(Supported by NSF MCB 9513439 to K. Kinnally). Apoptosis is integral to a wide range of biological processes. Recent findings from many laboratories suggest that mitochondria play a pivotal role in the apoptotic cascade in several cell types. For instance, apoptosis can be triggered in cells and cell-free systems by drugs that release proteins from mitochondria. This process can be blocked by bcl-2 and related apoptosis inhibitors that have been localized to mitochondria. Furthermore, in many cell types, the apoptosis-induced loss of mitochondrial membrane potential precedes hallmark events like loss of plasma membrane lipid asymmetry and nuclear changes such as chromatin condensation and DNA fragmentation. These observations suggest that changes in mitochondrial membrane permeability and organization may be integral, early steps in the apoptotic cascade. Experiments were initiated that will allow us to evaluate the effects of mechanically releasing discrete amounts of mitochondrial contents into living cells in the absence of agents (i.e., drugs) that may have pleiotropic effects. Preliminary work at the BMIRR reveals that just a single 5-7 ns pulse of 532 nm light from the laser microsurgery system is all that is needed to lyse a mitochondria within a living cell, and that 10-20 mitochondria can be lysed within a 1 min period. Preliminary data, which has formed the basis of a pending NIH grant, reveals that lysing just 10 of the 100-200 mitochondria within a cultured cell with this method appears to induce the cell to exit the cell cycle. Furthermore, since all the mitochondria in the irradiated cells eventually depolarized, there must also be an amplification of the laser-induced signal. Experiments are underway to determine whether the cells that exit the cell cycle ultimately die by the apoptotic or necrotic pathways. A timetable of onset of various markers is being established. Efforts are being made to visualize the wave of mitochondrial depolarization.